Aldefluor protocol to sort keratinocytes stem cells from skin

Author(s)

Ribeiro Noronha, Samuel Marcos ; Gragnani, Alfredo ; Calado Pereira, Thiago Antonio ; Alves Correa, Silvana Aparecida ; Bonucci, Jessica ; Ferreira, Lydia Masako

• Document type: Journal article
• Source: Acta Cirurgica Brasileira; v. 32, n. 11, p. 11-pg., 2017-11-01.

Abstract

Purpose: To investigate the use Aldefluor r and N, N-Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor r aliquots were prepared and maintained at temperature between 2 to 8 degrees C, or stored at -20 degrees C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor r 1.5 mu l and DEAB 15 mu l for 1 x 10(6) cells, incubated at 37 degrees C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor r has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol. (AU)

• FAPESP's process: 13/10905-0 - Keratinocyte growth factor in gene expression of immune system and wound healing in stem cells of the skin from burned patients
• Grantee: Alfredo Gragnani Filho
• Support Opportunities: Regular Research Grants